The present invention relates to chromosome painting and more particularly the fluorescent probes which can be used in methods such as the FISH (xe2x80x9cFluorescence In Situ Hybridizationxe2x80x9d) method. The invention also relates to combinations of fluorophores and of optical filters.
In situ hybridization is a technique which makes it possible to detect a DNA (or RNA) sequence by means of a probe having a specific sequence which is homologous to that studied. It is based on the complementarity of the nucleotides (A/T, A/U, G/C) and it can be carried out under precise physicochemical conditions on chromosome or tissue preparations. The result of the in situ hybridization process is the formation of a hybrid between a probe and a target. In situ hybridization includes a denaturing step and also a step for detecting the hybrid or the probe which is carried out after the in situ hybridization of the probe to the target. The sample may adhere in the form of a layer to the surface of the slide and this sample may, for example, comprise or contain individual chromosomes or chromosomal regions which have been treated in order to maintain their morphologies under denaturing conditions. In the context of fluorescence in situ hybridization, the probes are labeled with a fluorophore and the hybridization is revealed by fluorescent labeling.
The recent development of this technique allows the simultaneous visualization, on the same preparation, of several probes each revealed by a different fluorophore. This technique, called multicolor FISH or multi-FISH, has been made possible by the combination of filters specific for the wavelengths of emission of the different fluorescent molecules ensuring the labeling with the aid of a computer-aided imaging carried out by means of infrared-sensitive high-resolution cold CCD cameras (Schrxc3x6ck et al., 1996; Speicher et al., 1996).
The use of probes having a specific sequence homologous to a precise chromosomal sequence or a whole chromosome coupled with the potential for a multicolor fluorescent labeling makes it possible to develop so-called chromosome painting techniques, that is to say to obtain chromosomes of different colors and thus to obtain, if desired, a multicolor complete karyotype. Karyotype is understood to mean the characteristic arrangement of the chromosomes of a cell in the metaphase.
Within the general meaning of the term, xe2x80x9clabelingxe2x80x9d is understood to mean an entity such as a radioactive isotope or a nonisotopic entity such as enzymes, biotin, avidin, steptavidin, digoxygenin, luminescent agents, dyes, haptens and the like. The luminescent agents, depending on the source of excitation energy, may be classified into radioluminescent, chemoluminescent, bioluminescent and photoluminescent (including fluorescent and phosphorescent) agents. The term xe2x80x9cfluorescentxe2x80x9d refers in general to the property of a substance (such as a fluorophore) to produce light when it is excited by an energy source such as ultraviolet light for example. xe2x80x9cChromosomal paint probexe2x80x9d is understood to mean a probe or a probe composition such as the probe composition of this invention, which is suitable for hybridizing, under hybridization conditions, with a target which comprises a predetermined chromosome of a multichromosomal genome. If only a fraction of such a chromosome is present in the sample being subjected to such a hybridization with such a probe composition, then this fraction hybridizes and is identified. In practice, a painted probe of this invention may be mixed with a second, a third, and the like, so as to allow the labeling and the simultaneous detection of the two, three, and the like, predetermined chromosomes. The visualization of all the 24 human chromosomes has been made possible by the use of a labeling with a combination of fluorochromes. For example, in the case of the use of 5 different fluorophores, 31 combinations of fluorophores can be obtained. By using this labeling principle and 24 DNA probes specific for each of the human chromosomes, it has been possible to visualize each chromosome differentially. The attribution, by computer processing, of artificial colors to each of the combinations of fluorophores thus makes it possible to color the 24 human chromosomes differently.
Rapidly, the strong potentials of such a multicolor labeling have allowed the analysis of chromosomal aberrations which were difficult to detect up until now by conventional cytogenetic techniques for labeling chromosomes in bands (Summer et al., 1971; Dutrillaux and Lejeune, 1971) (Giema staining, labeling with BrdU, and the like). The principle of labeling of chromosomes in bands is based on the differences in the average base pair composition (richness in GC) between the bands and on the differences in the compaction of the chromatin between the chromosome bands. Chromosome painting has proved to be a very useful tool for detecting interchromosomal aberrations such as translocations, amplified DNA sequences such as the homogeneously stained regions called HSR (HSR for Homogeneously Staining Regions) or the excesses of chromosomal materials such as the marker chromosomes or double-minute chromosomes. Intrachromosomal aberrations such as deletions and duplications will only be detected as a function of the size of the aberrations, if the latter affect the length of the chromosomes, whereas chromosomal inversions will not at all be detectable by this method.
The limits of the use of the current spectral karyotyping as such are due to the fact that it does not make it possible to detect the nature of the chromosome bands involved in an inter- or intrachromosomal rearrangement. To do this, it is essential to couple this technique to the more conventional one of chromosome bands (R or G labeling) such as DAPI counterstaining, Giemsa or propidium iodide staining for example.
The requirement to combine different techniques of course constitutes a handicap in the analysis of chromosomal aberrations and, moreover, the use of the FISH or multi-FISH method which combines the high cost of the apparatus and the instrumentation necessary for the visualization of chromosome painting with the high cost of the probes specific for the chromosomes restricts the possibilities of this technique spreading to research laboratories or to diagnostic laboratories.
Paint probes currently available on the market (GIBCO-BRL, Oncor, Boehringer Manheim and the like) are obtained by DOP-PCR amplification using degenerate PCR primers of chromosomes or for fragments of chromosomes isolated by cumbersome techniques such as chromosome sorting by flow cytometry or microdissection of chromosomes. The hybridization of probes obtained by DOP-PCR does not generate chromosome bands on the chromosomes. The generation of chromosome bands was sought through the creation of artificial bands along chromosomes. This creation of artificial bands requires the use of cumbersome and expensive techniques. Furthermore, it results in bands which are not known reference marks in the field of cytogenetics.
Some others have described the use of chromosome paint probes obtained by amplification of chromosomes by IRS-PCR (Interspersed Repeated Sequences) using primers specific for DNA sequences which are repeated and dispersed in the genome, such as the Alu and LINE sequences. The combined use of LINE and Alu PCR primers for the amplification of human chromosomes by ISR-PCR was previously proposed by Lichter et al., 1990. However, the labeling in R bands which was obtained by the latter does not make it possible to ensure complete painting covering all the regions of the genome, in particular the telomeric regions and certain G chromosome bands.
The object of the present invention is to provide chromosomal probes which can be obtained inexpensively and which, in addition, make it possible to cause quality chromosome bands to appear directly on chromosomes painted in their entirety.
To do this, the present invention relates to probes intended for the labeling of a chromosome, characterized in that they are composed of a set of DNA segments which are more represented in certain chromosome bands and which are obtained by IRS-PCR amplification from said chromosomes with the aid of primers specific for the Alu and LINE DNA sequences.
The term xe2x80x9cprobexe2x80x9d refers to a polynucleotide or a mixture of polynucleotides such that DNA segments or DNA sequences are chemically combined with labeled individual entities. Each of the polynucleotides constituting a probe is characteristically in single-stranded form at the time of hybridization to the target.
The term xe2x80x9cDNA fragmentxe2x80x9d, xe2x80x9cDNA segmentxe2x80x9d generally indicates only a portion of the polynucleotide or of a sequence present in a chromosome or a chromosome portion. A polynucleotide for example may be cut or fragmented into a multitude of segments or of fragments. A chromosome characteristically contains regions which have DNA sequences containing repeated DNA segments. The term xe2x80x9crepeatedxe2x80x9d refers to the fact that a particular DNA segment is present many times (at least twice), dispersed or otherwise in the genome. The so-called IRS-PCR method using primers which hybridize with the dispersed repeated sequences of the genome, such as, for example, the Alu or LINE sequences.
xe2x80x9cGenomexe2x80x9d designates the complete and unique copy of the genetic instructions of an organism encoded by the DNA of this organism. In the present invention, the particular genome considered is multichromosomal such that the DNA is distributed in the cell between several individual chromosomes. The human genome is composed of 23 pairs of chromosomes, of which an XX or XY pair determine the sex.
The term xe2x80x9cchromosomexe2x80x9d refers to the support for the genes carrying heredity in a living cell which is derived from chromatin and which comprises DNA and protein components (essentially histones). The conventional international system for identifying and numbering the chromosomes of the human genome is used here. The size of an individual chromosome may vary within a multichromosomal genome and from one genome to another. In the present case of the human genome, the total length of DNA of a given chromosome is generally greater than 50 million bp. By way of comparison, the total length of the human genome is 3xc3x97109 bp.
The genome of mammals contains repeated DNA sequences dispersed over the whole genome. In humans, the majority of this type of sequences is represented by the different families of Alu sequences, which are about 106 in number and have in common a consensus sequence of 300 bp. The repeated LINES (or L1) sequences are, like the Alu sequences, widely distributed over the whole genome. They are nevertheless less numerous (about 104). Their consensus sequence is about 6 kb. They are preferably situated in the dark G bands (positive G or negative R), whereas the Alu sequences are instead situated in the dark R bands (positive R) (Korenberg and Kirowski, 1988).
The DNA segments amplified by IRS-PCR according to the invention may have as source somatic hybrids, preferably rodent-human somatic hybrids, chromosomes or fragments of chromosome. The chromosomes or fragments of chromosome may be obtained by chromosome sorting by flow cytometry or by chromosome microdissection. Within the framework of the preferred embodiment of the present invention, the DNA segments amplified by IRS-PCR have as source mono-chromosomal rodent-human somatic hybrids.
The DNA segments amplified by IRS-PCR according to the invention are more represented in one type of cytogenetic band. The preferred cytogenetic bands are the G bands or the R bands. In the preferred embodiment of the present invention, the DNA segments amplified by IRS-PCR are more represented in R bands.
Repeated sequences, analogous to the human repeated sequences, are also found in the genome of rodents. However, the divergence of this type of sequences between humans and rodents is sufficiently important for there to be little homology between them. This divergence allows a selective amplification of DNA segments contained in the human chromosome when human-rodent hybrids are used. Thus, starting with the DNA of a human-rodent somatic hybrid and using primers specific for the Alu and/or L1 consensus sequence, it is possible to selectively amplify by PCR the DNA sequences between two repeated sequences (xe2x80x9cinvertedxe2x80x9d position) separated by a distance  less than 5 kb. The product of amplification thus obtained consists of a set of fragments (whose size varies approximately from 100 bp to 5 kb) which is representative of practically the entire human chromosome contained in the DNA of the somatic hybrid.
In general, the present invention is of course more particularly intended for producing probes specific for human chromosomes, although it is possible to envisage chromosome paintings for other cell types (Sabile et al., 1997).
The probes of the present invention are characterized in that the probes are derived from a mixture of two IRS-PCR amplification products which is composed of:
PCR amplification product obtained using the primer specific for the Alu DNA sequences,
PCR amplification product obtained using the primer specific for the Alu DNA sequences and the primer specific for the LINE DNA sequences.
The present invention relates to a method of producing probes intended for labeling human chromosomes, characterized in that said method comprises the mixing of two amplification products obtained by two IRS-PCR amplifications from said chromosomes using, on the one hand, PCR primers specific for the Alu and LINE DNA sequences, and, on the other hand, PCR primers specific for the Alu DNA sequences.
Any primer specific for the Alu or LINE sequences may be used in the present invention. Preferably, the primers specific for the Alu DNA sequences consist of the SR1 primer whose sequence is described in SEQ ID No 1 and the primer specific for the LINE DNA sequence is preferably the L1H primer whose sequence is described in SEQ ID Nos 2 and 3.
Alternatively, probes according to the present invention may also be derived from a mixture of two IRS-PCR amplification products which is composed of:
PCR amplification product obtained using the primer specific for the LINE DNA sequences,
PCR amplification product obtained using the primer specific for the Alu DNA sequences and the primer specific for the LINE DNA sequences.
The present invention also relates to a method of producing probes intended for labeling human chromosomes, characterized in that said method comprises the mixing of two amplification products obtained by two IRS-PCR amplifications from said chromosomes using on the one hand PCR primers specific for the Alu and LINE DNA sequences, and, on the other hand, PCR primers specific for the LINE DNA sequences.
The present invention also comprises the use of fluorophores and of filters whose combination makes it possible to ensure a chromosome painting providing very readable karyotypes, that is to say to obtain contrasted and well-defined chromosome paint colors.
Thus, the DNA probes described above are labeled directly or indirectly by fluorescence techniques. Non-exhaustively, the fluorophores used for the labeling may be chosen from markers of the cyanine, rhodamine, fluorescein, Bobipy, Texas Red, Oregon Green, Cascade Blue type. In particular, all the fluorophores cited in xe2x80x9cHandbook of fluorescent probes and research chemicalsxe2x80x9d (Richard P Haugland, 1996, Molecular Probes, MTZ Spence Ed., more particularly p 145-146, 153, 155-156, 157-158, 161) can be used to label the probes of the present invention.
Preferably, the probes according to the invention are labeled with at least 1, 2, 3, 4 or 5 fluorophores chosen from the following group: fluorescein isothiocyanate (FITC), Texas Red (TR for Texas Red), cyanine 3 (Cy3), cyanine 5 (Cy5), cyanine 5.5 (Cy5.5), cyanine 7 (Cy7), Bodipy 630/650.
The preferred method for labeling the DNA probes is xe2x80x9cNick translationxe2x80x9d. However, the labeling may also be carried out by all the standard reactions for synthesis of DNA catalyzed by a polymerase and for labeling oligonucleotides. For example, the labeling may be carried out by the techniques of random priming, amplification or primer extension in situ.
The term xe2x80x9cdirectly labeled probexe2x80x9d designates or describes a nucleic acid probe whose labeling after the formation of hybrid with the target is detectable without subsequent reagent treatment of the hybrid. The probes using the FITC, Texas Red, Cy3 and Cy5 fluorophores according to the present invention are directly labeled.
The term xe2x80x9cindirectly labeled probexe2x80x9d designates or describes a nucleic acid probe whose labeling after the formation of hybrid with the target must undergo an additional reagent treatment with one or more reagents in order to combine therewith one or more entities from which a detectable compound finally result(s). For example, the probes may be labeled with DNP, digoxigenin or biotin and the revealing comprises bringing the probe into contact with an anti-DNP or anti-digoxigenin antibody labeled with a fluorophore or with an avidin coupled to a fluorophore. The probes using the fluorophores Cy7, Bodipy 630/650, Cy5.5. according to the present invention are indirectly labeled.
Preferably, the probe composition of the present invention comprises the largest possible number of xe2x80x9cdirectly labeledxe2x80x9d probes. In addition to the fact that the directly labeled probes are easier to use, they allow better resolution. This good resolution is important for good observation of the chromosome bands. Preferably, the probe composition of the present invention is of the xe2x80x9cdirect labelingxe2x80x9d type for all the fluorophores. A preferred probe composition of the present invention is of the xe2x80x9cdirect labelingxe2x80x9d type for 4 of the 5 fluorophores used. In another preferred composition, the directly labeled probes represent 3 probes out of 5 or 6 fluorophores used.
The present invention also relates to a set of probes intended for labeling human chromosomes, characterized in that it contains probes according to the present invention for each of the human chromosomes and for a number of them. This set of probes will make it possible to analyze in a single operation a complete karyotype so as to detect therein and to identify therein possible chromosomal aberrations as described above.
The present invention also relates to a multicolor FISH method intended for studying the karyotype, characterized in that the DNA probes are labeled with fluorophores and in that each fluorophore having a specific absorption and emission wavelength is combined with a pair of optical filteres, one for absorption and one for emission.
The fluorophores are chosen such that the overlapping of the absorption and emission spectra between the different fluorophores is minimal. More particularly, it is important that there is no overlapping between the absorption and emission maxima of the different fluorophores.
Each fluorophore is used with a pair of optical filters; one for absorption and one for emission. The filters make it possible to select the passbands such that the wavelengths corresponding to an overlap with another fluorophore are eliminated. Accordingly, the filters used in the present invention are preferably of the narrow passband optical filter type. The filters are preferably of a superior quality because it is important that the filter does not allow light outside the passband to pass through.
Preferably, the present invention also relates to a multicolor FISH method intended in particular for studying the karyotype, characterized in that the DNA probes according to the present invention are labeled with fluorophores and in that each fluorophore having a specific absorption and emission wavelength is combined with a pair of optical filters, one for absorption and the other for emission, said method using fluorophores and pairs of filters chosen from the following group:
a) the fluorophore FITC having a maximum absorption wavelength of 494 nm and a maximum emission wavelength of 517 nm combined with an excitation filter of the 490DF30 type (Omega Optical) and with an emission filter of the 530DF30 type (Omega Optical),
b) the fluorophore Cy3 having a maximum absorption wavelength of 554 nm and a maximum emission wavelength of 568 nm combined with an excitation filter of the 546DF10 type (Omega Optical) and with an emission filter of the 570DF10 type (Omega Optical),
c) the fluorophore TR having a maximum absorption wavelength of 593 nm and a maximum emission wavelength of 613 nm combined with an excitation filter of the 590DF10 type (Omega Optical) and with an emission filter of the 615DF10 type (Omega Optical),
d) the fluorophore Cy5 having a maximum absorption wavelength of 652 nm and a maximum emission wavelength of 670 nm combined with an excitation filter of the 650DF20 type (Omega Optical) and with an emission filter of the 670DF10 type (Omega Optical),
e) the fluorophore Cy7 having a maximum absorption wavelength of 743 nm and a maximum emission wavelength of 767 nm combined with an excitation filter of the 740DF25 type (Omega Optical) and with an emission filter of the 780EFLP type (Omega Optical),
f) the fluorophore Cy5.5 having a maximum absorption wavelength of 675 nm and a maximum emission wavelength of 694 nm combined with an excitation filter of the 680DF20 type (Omega Optical) and with an emission filter of the 700EFLP type (Omega Optical),
g) the fluorophore Bodipy 630/650 having a maximum absorption wavelength of 632 nm and a maximum emission wavelength of 658 nm combined with an excitation filter of the 630DF20 type (Omega Optical) and with an emission filter of the 650DF10 type (Omega Optical).
The invention also relates to a multicolor FISH diagnostic kit characterized in that it comprises DNA probes according to the present invention labeled with fluorophores and in that each fluorophore having a specific absorption and emission wavelength is combined with a pair of optical filters, one for absorption and one for emission, said kit using fluorophores and pairs of filters chosen from the following group:
a) the fluorophore FITC having a maximum absorption wavelength of 494 nm and a maximum emission wavelength of 517 nm combined with an excitation filter of the 490DF30 type (Omega Optical) and with an emission filter of the 530DF30 type (Omega Optical),
b) the fluorophore Cy3 having a maximum absorption wavelength of 554 nm and a maximum emission wavelength of 568 nm combined with an excitation filter of the 546DF10 type (Omega Optical) and with an emission filter of the 570DF10 type (Omega Optical),
c) the fluorophore TR having a maximum absorption wavelength of 593 nm and a maximum emission wavelength of 613 nm combined with an excitation filter of the 590DF10 type (Omega Optical) and with an emission filter of the 615DF10 type (Omega Optical),
d) the fluorophore Cy5 having a maximum absorption wavelength of 652 nm and a maximum emission wavelength of 670 nm combined with an excitation filter of the 650DF20 type (Omega Optical) and with an emission filter of the 670DF10 type (Omega Optical),
e) the fluorophore Cy7 having a maximum absorption wavelength of 743 nm and a maximum emission wavelength of 767 nm combined with an excitation filter of the 740DF25 type (Omega Optical) and with an emission filter of the 780EFLP type (Omega Optical),
f) the fluorophore Cy5.5 having a maximum absorption wavelength of 675 nm and a maximum emission wavelength of 694 nm combined with an excitation filter of the 680DF20 type (Omega Optical) and with an emission filter of the 700EFLP type (Omega Optical),
g) the fluorophore Bodipy 630/650 having a maximum absorption wavelength of 632 nm and a maximum emission wavelength of 658 nm combined with an excitation filter of the 630DF20 type (Omega Optical) and with an emission filter of the 650DF10 type (Omega Optical).
The filters according to the present invention are preferably such that:
they are of the 6-cavity type,
they have an ADI of 0xc2x0;
they have a tolerance xcex0xc2x120% of FWHM,
they have a tolerance on FWHM of xc2x120% of FWHM,
they have an OD5 out-of-passband rejection for UV at 1200 nm
they have a transmission curve Txe2x89xa750% at xcex0.
Preferably, the filters must also have a centered useful diameter greater than 21 mm, and a thickness xe2x89xa67 mm,
The fluorophores and the above filters may be used for the labeling of the probes according to the present invention or alternatively for different probes used, for example, for chromosome painting or for multicolor FISH.
In the present invention, xe2x80x9cfiltersxe2x80x9d is understood to mean narrow passband interference filters which transmit light within a given very narrow spectral band centered around the reference wavelength xcex0. They are characterized by their transmission curve: T=f(xcex). The width of the band is defined by the full width at half maximum transmission (FWHM for xe2x80x9cFull Width at Half Maximum transmissionxe2x80x9d).
Outside the passband, the filter allows a residual signal which is as attenuated as possible to pass through.
The interference filters function on the principle of constructive and destructive interference. The basic component of an interference filter is called cavity. It has two stacks of reflectors separated by a layer of a dielectric solid. The higher the number of cavities, the more rectangular the shape of the transmission curve (that is to say the greater the slope of this curve). Moreover, the higher the number of cavities, the better the coefficient of attenuation outside the passband.
For the multifluorescence application, the excitation or emission spectra of the fluorochromes used are very close to each other. It is therefore necessary to recover the minimum amount of signal possible outside the passband. Accordingly, 6-cavity filters which offer the best characteristics at this level were chosen.
The filters used preferably have the following specifications:
they are designed to be used at normal light incidence,
the tolerance on the centre wavelength (xcex0) is xc2x120% of the passband, for example, for a filter with a passband of 10 nm, xcex0 will be defined with a tolerance of xc2x12 nm,
the tolerance on the width of the passband is xc2x120%,
the coefficient of transmission T of these filters is greater than 50%,
the out-of-passband rejection of these filters is 5OD for ultraviolet at 1200 nm; this means that outside the passband, the coefficient of transmission is 10xe2x88x925, that is to say 0.001%. For standard filters, the out-of-passband rejection is ensured for wavelengths ranging from 0.8 xcex0 to 1.2 xcex0; for example, for a filter of xcex0=620 nm, the out-of-passband rejection occurs only between 500 and 740 nm. However, for the multifluorescence application, fluorochromes are observed whose spectra extend from 350 to 800 nm. Accordingly, filters were used whose out-of-passband rejection is ensured for ultraviolet at 1200 nm.
Finally, the present invention relates to a labeling kit characterized in that it comprises at least DNA probes as described above or a set of probes as mentioned above.
The present invention relates to a multicolor FISH diagnostic kit, characterized in that it comprises the DNA probes as described above or a set of DNA probes as mentioned above and a combination of filters and of fluorophores as described above.
The FISH or multi-FISH technique to which reference is or will be made several times in the present description is in particular described in Speicher et al., 1996; Schrxc3x6ck et al., 1996.
Other characteristics and advantages of the present invention will emerge on reading the examples below.
The combinations of fluorophores and of optical filters described in the invention may be used in multiple techniques involving fluorescence microscopy. Indeed, the fluorophores described in the present invention may be used to label many molecules or structures. Nonexhaustively, said fluorophores may be used to label polypeptides, antibodies, nucleic acids, phospholipids, fatty acids, sterol derivatives, membranes, organelles and many other biological macromolecules. The organelles may be mitochondria, endoplasmic reticulum, Golgi apparatus and lysosomes.
The combination of fluorophores and of optical filters according to the invention may be used to carry out FISH. In particular, this may allow the simultaneous use of several probes. This combination may be used to study multiple aspects such as cell morphology, the cytoskeleton, cell receptors, ion channels, neurotransmitters, the circulation of fluids, membrane fluidity, cell viability and proliferation, apoptosis, pinocytosis, endocytosis and exocytosis, transduction, pH and ion concentrations (for example calcium, potassium, magnesium and zinc concentrations) (Richard P Haugland, 1996, Molecular Probes, MTZ Spend Ed.). It may allow the study of expression and translation.
The invention therefore also relates to a combination of fluorophores chosen from: fluorescein isothiocyanate (FITC), Texas Red (TR for Texas Red), cyanine 3 (Cy3), cyanine 5 (Cy5), cyanine 5.5 (Cy5.5), cyanine 7 (Cy7), Bodipy 630/650.
Preferably, the combination of fluorophores comprises at least 2, 3, 4, 5, 6 or 7 fluorophores chosen from: fluorescein isothiocyanate (FITC), Texas Red (TR for Texas Red), cyanine 3 (Cy3), cyanine 5 (Cy5), cyanine 5.5 (Cy5.5), cyanine 7 (Cy7), Bodipy 630/650.
A combination of preferred fluorophores of the present invention comprises the following 5 fluorophores: fluorescein isothiocyanate (FITC), Texas Red (TR), cyanine 3 (Cy3), cyanine 5 (Cy5) and cyanine 7 (Cy7).
Another preferred combination of fluorophores of the present invention comprises the following 6 fluorophores: fluorescein isothiocyanate (FITC), Texas Red (TR), cyanine 3 (Cy3), Bodipy 630/650, cyanine 5 (Cy5) and cyanine 7 (Cy7).
Another preferred combination of fluorophores of the present invention comprises the following 6 fluorophores: fluorescein isothiocyanate (FITC), Texas Red (TR), cyanine 3 (Cy3), Bodipy 630/650 and cyanine 5 (Cy5).
The combinations of fluorophores according to the invention may be included in a multicolor FISH diagnostic kit.
The combinations of fluorophores according to the invention may be used to label an entity chosen from polypeptides, antibodies, nucleic acids, phospholipids, fatty acids, sterol derivatives, membranes, organelles and biological macromolecules.
In addition, the invention relates to a combination of fluorophores combined with a pair of optical filters chosen from:
a. the fluorophore FITC having a maximum absorption wavelength of 494 nm and a maximum emission wavelength of 517 nm combined with an excitation filter of the 490DF30 type (Omega Optical) and with an emission filter of the 530DF30 type (Omega Optical),
b. the fluorophore Cy3 having a maximum absorption wavelength of 554 nm and a maximum emission wavelength of 568 nm combined with an excitation filter of the 546DF10 type (Omega Optical) and with an emission filter of the 570DF10 type (Omega Optical),
c. the fluorophore TR having a maximum absorption wavelength of 593 nm and a maximum emission wavelength of 613 nm combined with an excitation filter of the 590DF10 type (Omega Optical) and with an emission filter of the 615DF10 type (Omega Optical),
d. the fluorophore Cy5 having a maximum absorption wavelength of 652 nm and a maximum emission wavelength of 670 nm combined with an excitation filter of the 650DF20 type (Omega Optical) and with an emission filter of the 670DF10 type (Omega Optical),
e. the fluorophore Cy7 having a maximum absorption wavelength of 743 nm and a maximum emission wavelength of 767 nm combined with an excitation filter of the 740DF25 type (Omega Optical) and with an emission filter of the 780EFLP type (Omega Optical),
f. the fluorophore Cy5.5 having a maximum absorption wavelength of 675 nm and a maximum emission wavelength of 694 nm combined with an excitation filter of the 680DF20 type (Omega Optical) and with an emission filter of the 700EFLP type (Omega Optical),
g. the fluorophore Bodipy 630/650 having a maximum absorption wavelength of 632 nm and a maximum emission wavelength of 658 nm combined with an excitation filter of the 630DF20 type (Omega Optical) and with an emission filter of the 650DF10 type (Omega Optical).
Preferably, the combination of fluorophores combined with a pair of optical filters comprises at least 1, 2, 3, 4, 5, 6 or 7 fluorophores and filters chosen from:
a) the fluorophore FITC having a maximum absorption wavelength of 494 nm and a maximum emission wavelength of 517 nm combined with an excitation filter of the 490DF30 type (Omega Optical) and with an emission filter of the 530DF30 type (Omega Optical),
b) the fluorophore Cy3 having a maximum absorption wavelength of 554 nm and a maximum emission wavelength of 568 nm combined with an excitation filter of the 546DF10 type (Omega Optical) and with an emission filter of the 570DF10 type (Omega Optical),
c) the fluorophore TR having a maximum absorption wavelength of 593 nm and a maximum emission wavelength of 613 nm combined with an excitation filter of the 590DF10 type (Omega Optical) and with an emission filter of the 615DF10 type (Omega Optical),
d) the fluorophore Cy5 having a maximum absorption wavelength of 652 nm and a maximum emission wavelength of 670 nm combined with an excitation filter of the 650DF20 type (Omega Optical) and with an emission filter of the 670DF10 type (Omega Optical),
e) the fluorophore Cy7 having a maximum absorption wavelength of 743 nm and a maximum emission wavelength of 767 nm combined with an excitation filter of the 740DF25 type (Omega Optical) and with an emission filter of the 780EFLP type (Omega Optical),
f) the fluorophore Cy5.5 having a maximum absorption wavelength of 675 nm and a maximum emission wavelength of 694 nm combined with an excitation filter of the 680DF20 type (Omega Optical) and with an emission filter of the 700EFLP type (Omega Optical),
g) the fluorophore Bodipy 630/650 having a maximum absorption wavelength of 632 nm and a maximum emission wavelength of 658 nm combined with an excitation filter of the 630DF20 type (Omega Optical) and with an emission filter of the 650DF10 type (Omega Optical).
The combinations of fluorophores combined with the pair of optical filters according to the invention may be included in a multicolor FISH diagnostic kit.
The combinations of fluorophores combined with a pair of optical filters according to the invention may be used to label an entity chosen from polypeptides, antibodies, nucleic acids, phospholipids, fatty acids, sterol derivatives, membranes, organelles and biological macromolecules.
The probes and combinations of fluorophores according to the invention may be used with any type of microscope (monochromator, laser, fluorescence microscope). Preferably, the invention uses a fluorescence microscope.
Various publications and patents are cited in the description. The disclosures contained in the publications and patents identified by references in this application are incorporated by way of reference into the present application for a more detailed description of the content of the present invention.